Structural Characterization of Organic Multilayers on Silicon(111

Sep 8, 2004 - A lower yield of approximately 30% for the coupling of SMP to ..... The authors acknowledge the assistance of Ian Gentle and Jeremy Rugg...
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Structural Characterization of Organic Multilayers on Silicon(111) Formed by Immobilization of Molecular Films on Functionalized Si-C Linked Monolayers Till Bo¨cking,*,†,‡ Michael James,§ Hans G. L. Coster,† Terry C. Chilcott,† and Kevin D. Barrow‡ UNESCO Centre for Membrane Science and Technology, Department of Biophysics, School of Physics, University of New South Wales, Sydney, NSW 2052, Australia, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia, and The Bragg Institute, Australian Nuclear Science and Technology Organisation (ANSTO), Lucas Heights Research Laboratory, Lucas Heights, NSW 2234, Australia Received June 21, 2004. In Final Form: July 21, 2004 Silicon(111)-H surfaces were derivatized with ω-functionalized alkenes in UV-mediated and thermal hydrosilylation reactions to give Si-C linked monolayers. Additional molecular layers of organic compounds were coupled either directly or via linker molecules to the functionalized alkyl monolayers. In the first instance, amino-terminated monolayers were prepared from a tert-butoxycarbonyl-protected ω-aminoalkene followed by removal of the protecting group. Various thiols were coupled to the monolayer using a heterobifunctional linker, which introduced maleimide groups onto the surface. In the second system, N-hydroxysuccinimide (NHS) ester-terminated monolayers were formed by reaction of Si-H with N-succinimidyl undecenoate. The reactivity of the NHS ester groups was confirmed by further modification of the monolayer. The stepwise assembly of these multilayer structures was characterized by X-ray reflectometry and X-ray photoelectron spectroscopy.

Introduction The controlled immobilization of chemical and biological molecules on surfaces is of great importance for the preparation of biosensors, as well as chemical, protein and DNA microarrays, with applications in diagnostics, drug discovery, proteomics, genomics and high-throughput screening of molecular interactions.1-8 To improve the stability and sensitivity of biological and chemical arrays, or “chips”, it is advantageous to ensure that molecularcapture agents are covalently linked to the surface in a well-defined orientation and density. Functionalized selfassembled monolayers (SAMs) of thiols on gold or organosilanes on silica surfaces have been used extensively for the controlled immobilization of biomolecules. Recent examples include the preparation of peptide and carbohydrate biochips on SAMs on gold,9 attachment of DNA oligomers to silanized slides10 or mixed alkanethiols on gold for microarrays,11 and the coupling of protein to porous * Author to whom correspondence should be addressed. Email: [email protected]. † Department of Biophysics, UNSW. ‡ School of Biotechnology and Biomolecular Sciences, UNSW. § The Bragg Institute, ANSTO. (1) Lam, K. S.; Renil, M. Curr. Opin. Chem. Biol. 2002, 6, 353-358. (2) Emili, A. Q.; Cagney, G. Nat. Biotechnol. 2000, 18, 393-397. (3) Lal, S. P.; Christopherson, R. I.; dos Remedios, C. G. Drug Discovery Today 2002, 7, S143-S149. (4) Keusgen, M. Naturwissenschaften 2002, 89, 433-444. (5) Angenendt, P.; Glokler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309, 253-260. (6) Cahill, D. J. J. Immunol. Methods 2001, 250, 81-91. (7) Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827-836. (8) MacBeath, G. Nat. Genet. 2002, 32, 526-532. (9) Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Langmuir 2003, 19, 1522-1531. (10) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031-3039. (11) Riepl, M.; Enander, K.; Liedberg, B.; Schaeferling, M.; Kruschina, M.; Ortigao, F. Langmuir 2002, 18, 7016-7023.

silicon for the development of an optical biosensor.12 A disadvantage associated with thiol SAMs on gold is the limited stability of the gold-thiol bond. Although organosilane SAMs on silicon dioxide surfaces exhibit higher stability, degradation of the surface is still possible because the Si-O-C bond is prone to hydrolysis in aqueous media, especially under alkaline conditions. Surface modification of silicon with highly robust Si-C linked organic monolayers is currently an area of intensive research not only because of its technological applications for passivating or bio-functionalizing semiconductor devices but also for the study of chemical and electronic processes at the semiconductor surface.13-18 Consequently, numerous synthetic strategies have been developed over the past few years to form methyl-terminated and functionalized monolayers on silicon. Alkenes can be reacted with hydride-terminated, flat silicon surfaces to form highly ordered Si-C linked monolayers using a variety of methods. These include photochemical hydrosilylation,19-23 thermally induced hydrosilylation,24-27 free(12) Dancil, K.-P. S.; Greiner, D. P.; Sailor, M. J. J. Am. Chem. Soc. 1999, 121, 7925-7930. (13) Buriak, J. M. Chem. Rev. 2002, 102, 1271-1308. (14) Stewart, M. P.; Buriak, J. M. Comments Inorg. Chem. 2002, 23, 179-203. (15) Stewart, M. P.; Buriak, J. M. Adv. Mater. 2000, 12, 859-869. (16) Buriak, J. M. Chem. Commun. 1999, 1051-1060. (17) Wayner, D. D. M.; Wolkow, R. A. J. Chem. Soc., Perkin Trans. 2 2002, 23-34. (18) Sieval, A. B.; Linke, R.; Zuilhof, H.; Sudholter, E. J. R. Adv. Mater. 2000, 12, 1457-1460. (19) Barrelet, C. J.; Robinson, D. B.; Cheng, J.; Hunt, T. P.; Quate, C. F.; Chidsey, C. E. D. Langmuir 2001, 17, 3460-3465. (20) Cicero, R. L.; Linford, M. R.; Chidsey, C. E. D. Langmuir 2000, 16, 5688-5695. (21) Boukherroub, R.; Wayner, D. D. M. J. Am. Chem. Soc. 1999, 121, 11513-11515. (22) Boukherroub, R.; Morin, S.; Bensebaa, F.; Wayner, D. D. M. Langmuir 1999, 15, 3831-3835.

10.1021/la048474p CCC: $27.50 © 2004 American Chemical Society Published on Web 09/08/2004

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Figure 1. Preparation of Si-C linked alkyl monolayers on the silicon surface: (a) Removal of the silicon dioxide layer by etching in HF or NH4F solution yields a hydride terminated surface which is (b) reacted with terminal olefins in a UV-light-mediated, thermal, or catalyzed reaction.

radical initiation,24,28,29 and Lewis acid catalyzed reactions.22 UV-mediated and thermal methods are convenient to use and are shown in Figure 1. The proposed reaction mechanism for the formation of Si-C linked monolayers is a surface-propagated radical chain reaction, which is initiated by UV light or heat-mediated formation of a silyl radical (dangling bond).30,31 The silyl radical then reacts with an alkene, leading to the formation of a carboncentered radical, which may then abstract a hydrogen from a neighboring Si-H site to create a new reactive dangling bond (propagation of the radical chain reaction).30,31 The resulting Si-C linked monolayers are extremely robust and withstand harsh chemical treatment with boiling aqueous and organic solvents, aqueous HF, and KOH,32,33 as well as temperatures up to 615 K.34 This chemistry is capable of producing nearly oxide-free surfaces with a low density of surface states which opens the possibility of integrating the detection of binding events at the silicon surface with changes in the electronic structure of the semiconductor. A further advantage of using silicon as a substrate is that the organic or biological molecular systems can be integrated with existing semiconductor microfabrication technology. Numerous approaches have been taken to introduce reactive headgroup functionalities onto the Si-C linked monolayers. Chidsey and co-workers prepared aminereactive surfaces by free-radical chlorosulfonation of octadecyl monolayers35 and recently demonstrated the gasphase chlorination with Cl2.29 Free amino groups are incompatible with UV- and heat-mediated hydrosilylation reactions and must therefore be protected, for example, with phthalimide, acetamide,27 or tert-butoxycarbonyl36,37 groups. Deprotected amino-terminated monolayers have (23) Effenberger, F.; Gotz, G.; Bidlingmaier, B.; Wezstein, M. Angew. Chem., Int. Ed. 1998, 37, 2462-2464. (24) Linford, M. R.; Fenter, P.; Eisenberger, P. M.; Chidsey, C. E. D. J. Am. Chem. Soc. 1995, 117, 3145-3155. (25) Sieval, A. B.; Demirel, A. L.; Nissink, J. W. M.; Linford, M. R.; van der Maas, J. H.; de Jeu, W. H.; Zuilhof, H.; Sudhoelter, E. J. R. Langmuir 1998, 14, 1759-1768. (26) Sieval, A. B.; Vleeming, V.; Zuilhof, H.; Sudhoelter, E. J. R. Langmuir 1999, 15, 8288-8291. (27) Sieval, A. B.; Linke, R.; Heij, G.; Meijer, G.; Zuilhof, H.; Sudhoelter, E. J. R. Langmuir 2001, 17, 7554-7559. (28) Linford, M. R.; Chidsey, C. E. D. J. Am. Chem. Soc. 1993, 115, 12631-12632. (29) Linford, M. R.; Chidsey, C. E. D. Langmuir 2002, 18, 62176221. (30) Cicero, R. L.; Chidsey, C. E. D.; Lopinski, G. P.; Wayner, D. D. M.; Wolkow, R. A. Langmuir 2002, 18, 305-307. (31) Lopinski, G. P.; Wayner, D. D. M.; Wolkow, R. A. Nature 2000, 406, 48-51. (32) Boukherroub, R.; Morin, S.; Wayner, D. D. M.; Bensebaa, F.; Sproule, G. I.; Baribeau, J. M.; Lockwood, D. J. Chem. Mater. 2001, 13, 2002-2011. (33) Boukherroub, R.; Wayner, D. D. M.; Lockwood, D. J.; Canham, L. T. J. Electrochem. Soc. 2001, 148, H91-H97. (34) Sung, M. M.; Kluth, G. J.; Yauw, O. W.; Maboudian, R. Langmuir 1997, 13, 6164-6168. (35) Wagner, P.; Nock, S.; Spudich, J. A.; Volkmuth, W. D.; Chu, S.; Cicero, R. L.; Wade, C. P.; Linford, M. R.; Chidsey, C. E. D. J. Struct. Biol. 1997, 119, 189-201. (36) Lin, Z.; Strother, T.; Cai, W.; Cao, X.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 788-796. (37) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535-3541.

been used for the immobilization of DNA oligonucleotides on the surface.37,38 Thermal hydrosilylation of neat, unprotected undecenoic acid at 200 °C also results in disordered monolayers since the carboxylic acid group reacts with the Si-H surface to form siloxane esters.25 To produce well-ordered monolayers with acid functionality using UV-mediated or thermal reactions at high temperature, the Si-H surface can be reacted with carboxylic acids protected as esters followed by ester hydrolysis.21,25,38 Recently it was shown that, at moderate temperature (95 °C), undecenoic acid can also be covalently attached to the Si-H surface in a thermal hydrosilylation reaction without considerable side-reaction of the free carboxylic acid group with the Si-H surface.39 In either case, the resulting acid-terminated surface can be subsequently activated with a carbodiimide and N-hydroxysuccinimide (NHS) for the reaction with nucleophiles. Alternatively, reactive esters of alkenes can be synthesized prior to attachment to the silicon surface. Wojtyk et al.40 showed that the derivatization of porous silicon surfaces with solutions of N-succinimidyl undecenoate or S-ethyl undecenoate leads to the formation of oxide-free, activated acid monolayers which can subsequently be reacted with amines or terminal cysteine residues, respectively, to form amide linkages. The alkene with a functional group can be diluted with an alkene with a different terminal group so that the density of activated esters and, therefore, the points of attachment for biological molecules can be controlled. In this study, we have prepared multilayer structures by attachment of organic model compounds to Si-C linked ω-functionalized alkyl monolayers on the Si(111) surface. Monolayers with two different terminal functional groups were used as base layers for the immobilization of molecules. The first approach is based on an aminoterminated surface and the second approach on a surface terminated with NHS ester groups. The quality, chemical composition, and structure of the surfaces was characterized by X-ray photoelectron spectroscopy (XPS) and X-ray reflectivity (XR) after each modification step. A detailed understanding of the reactions occurring at the monolayer surfaces and the resulting structures of the different layers will aid the development of improved immobilization strategies for devices with improved sensitivity and stability. Experimental Methods Materials. All chemicals were reagent grade or higher and used as received unless stated otherwise. MilliQ water (18 MΩ cm) was used for the rinsing of samples and the preparation of solutions. Ethanol, methanol, hexane, and dichloromethane were redistilled. The chemicals used for cleaning and etching of silicon wafer pieces (30% H2O2, 98% H2SO4 and 40% NH4F solution) were of SEMI (Olin), CMOS (Baker), or Finyte (Baker) grade. (38) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205-1209. (39) Boukherroub, R.; Wojtyk, J. T. C.; Wayner, D. D. M.; Lockwood, D. J. J. Electrochem. Soc. 2002, 149, H59-H63. (40) Wojtyk, J. T. C.; Tomietto, M.; Boukherroub, R.; Wayner, D. D. M. J. Am. Chem. Soc. 2001, 123, 1535-1536.

Characterization of Organic Multilayers on Si(111)

Langmuir, Vol. 20, No. 21, 2004 9229 2 min, blown dry under nitrogen, and packaged under argon in glass vials for transport. X-ray reflectivity curves were acquired using a Bruker D8 Advance diffractometer in reflectometer mode. Cu KR (λ ) 1.54056 Å) radiation produced from a (40 kV) tube source was focused with a Go¨bel mirror, collimated with preand post-sample slits, and detected using a YAP scintillation detector. Reflectivity data are presented as a function of momentum transfer Qz ) (4π sin θ)/λ, where λ is the wavelength and θ is the angle of incidence of the X-ray beam onto the sample. Contrast in XR depends on the changes in electron density Fel(z) of the material normal to the surface. Structural parameters such as the thickness (d), electron density (Fel), and interfacial roughness (σ) of the individual layers of a stratified sample may be determined by refining a structural model for the reflectivity data. The Parratt32 software43 was used to fit model parameters to measured sets of XR data with linear background correction. The reflectivity calculations in Parratt32 are carried out according to the dynamical approach by Parratt44 based on stratified media. The Parratt32 program includes roughnesses in the Fresnel coefficient, as suggested by Ne´vot and Croce.45 In the fitting routine, the scattering length density of the substrate (silicon) was fixed at an appropriate value (0.80 e- A-3). A slab model consisting of a homogeneous layer with an estimated scattering length density, thickness, and roughness for the organic film was then refined to fit the measured XR data. Additional slabs were introduced into the model for samples derivatized with multiple organic layers or heterogeneous monolayers when a single layer model did not yield a satisfactory fit to the measured XR data.

Preparation of Si-C Linked Monolayers. Si(111) wafers (n-type, 2-8 Ω cm or p-type, 1-10 Ω cm) were cleaved into pieces (approximately 10 × 15 mm2) and cleaned in concentrated H2SO4 :30% H2O2 (3:1, v/v) at 90 °C for 20-30 min followed by copious rinsing with MilliQ water. Caution: Acidic solutions of concentrated hydrogen peroxides react violently with organic materials and should be handled with extreme care. Hydrogenterminated Si(111) surfaces were prepared by etching in a deoxygenated 40% solution of NH4F for 15-20 min. The 40% NH4F solution was deoxygenated by bubbling with argon for at least 30 min. Under these conditions, atomically flat Si(111)-H surfaces can be obtained.41,42 Most samples were briefly rinsed with MilliQ water. Formation of monolayers by attachment of alkenes was achieved using three different methods: photochemical reaction, thermal reaction in neat alkene, and thermal reaction in refluxing mesitylene. For photochemical hydrosilylation reactions, alkenes were used neat or as solutions in mesitylene and were deoxygenated by bubbling with argon for at least 1 h. The freshly etched silicon wafer piece was placed into a Teflon holder inside a custommade reaction chamber with a quartz glass window. The chamber was evacuated to < 0.1 mmHg and backfilled with argon several times. The alkene was injected onto the Si-H terminated sample inside the chamber such that it was covered with a thin film. After irradiation with UV light for 2-3 h, the chamber was opened to the atmosphere and the sample was rinsed with solvents as described below. The thermal reaction in neat alkenes was carried out as follows. The neat alkene was placed into a Schlenk flask and degassed by five freeze-pump-thaw cycles. After transfer of the freshly etched silicon wafer piece to the Schlenk flask, the flask was immersed in an oil bath at 95 °C for 16-18 h. Then the flask was opened to the atmosphere, and the sample was rinsed as described below. Thermal hydrosilylation in refluxing mesitylene was carried out as described by Sieval et al.26 The freshly etched silicon wafer piece was immersed in a deoxygenated solution of the alkene in mesitylene kept under a nitrogen atmosphere. The solution was refluxed for 2-3 h while a slow stream of nitrogen was bubbled through the solution. Derivatized silicon samples were rinsed several times with hexane, dichloromethane, tetrahydrofuran, and ethanol and blown dry under a stream of nitrogen. Derivatization of Amino-Terminated Surfaces. The tertbutoxycarbonyl (t-Boc) protecting group was removed by treatment of the sample with 25% TFA in dichloromethane for 60 min.37 This was followed by immersion in 10% NH4OH for 2-3 min to obtain a surface terminated with unprotonated amino groups.37 In the next step, the heterobifunctional linker Nsuccinimidyl 3-maleimidopropionate (SMP) was coupled to the terminal, free amino groups of the alkyl monolayer to introduce thiol-reactive maleimide groups onto the surface. The wafer was immersed in a 25 mM solution of SMP in dimethylformamide:25 mM NaH2PO4, pH 8.0 (1:4, v/v) for 30 min. After being rinsed with dimethylformamide, water, and methanol, the sample was blown dry under nitrogen. Thiol addition to maleimide groups was carried as follows. The silicon wafer with surface maleimide groups was immersed in a solution of 10-100 mM thiol in methanol containing triethylamine for 40 min to 2 h followed by rinsing with methanol. Derivatization of NHS Ester-Terminated Surfaces. 4-(Trifluoromethyl)benzylamine was linked to the surface in dichloromethane containing N,N-diisopropylethylamine. The sample was rinsed with methanol and dichloromethane and blown dry under nitrogen. XPS Measurements. XP spectra were obtained using an EscaLab 220-IXL spectrometer with a monochromated Al KR source (1486.6 eV), hemispherical analyzer, and multichannel detector. The spectra were accumulated at a takeoff angle of 90° with a 0.79 mm2 spot size at a pressure of less than 10-8 mbar. X-Ray Reflectivity Measurements. Samples for X-ray reflectometry (XR) were cleaned in boiling dichloromethane for

In this study, we have characterized the multilayer structures formed by covalent attachment of organic model compounds to ω-functionalized alkyl monolayers on the silicon surface with two different terminal functional groups and coupling chemistries: (1) thiol binding to amino-terminated surfaces via a heterobifunctional linker and (2) amine binding to surfaces terminated with NHS ester groups. Strategy for the Coupling of Thiols to AminoTerminated Surfaces. The approach taken for formation of amino-terminated alkyl layers and the immobilization of thiols via heterobifunctional linkers was based on the methods developed by Strother et al.37 and is shown in Figure 2. N-tert-Butoxycarbonyl-10-undecenamine was reacted with the freshly prepared Si-H surface in a UVmediated hydrosilylation reaction to form a t-Boc-protected undecanamine monolayer (surface 1). After removal of the t-Boc group with 25% trifluoroacetic acid (TFA) in dichloromethane, the amino-terminated surface (surface 2) was modified with the heterobifunctional linker SMP, which coupled via its activated NHS ester to the amino groups and introduced thiol-reactive maleimide groups onto the surface (surface 3). Sulfhydryl-containing molecules (shown in Figure 2b) were reacted with the surfaceimmobilized maleimide groups to form stable thioether linkages (surface 4). Chemical and structural changes on the surface after each step were monitored by XPS and X-ray reflectometry. The binding of thiols to surfaceimmobilized maleimide groups was also analyzed by using sulfhydryl-containing radioactively labeled probes. Preparation and XPS Characterization of t-BocProtected Amino-Terminated Monolayers. Modification of the silicon surface with t-Boc undecenamine was attempted in photochemical and thermal hydrosilylation reactions. Figure 3 shows the XP survey spectrum of a Si(111) surface derivatized with a monolayer of t-Boc-

(41) Allongue, P.; Henry de Villeneuve, C.; Morin, S.; Boukherroub, R.; Wayner, D. D. M. Electrochim. Acta 2000, 45, 4591-4598. (42) Wade, C. P.; Chidsey, C. E. D. Appl. Phys. Lett. 1997, 71, 16791681.

(43) Braun, C. Parratt32, version 1.5.2; Neutron Scattering Center, Hahn Meitner Institut: Berlin, 1999. (44) Parratt, L. G. Phys. Rev. 1954, 95, 359-369. (45) Nevot, L.; Croce, P. Rev. Phys. Appl. 1980, 15, 761-779.

Results and Discussion

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Figure 4. XP narrow spectra of the carbon 1s region of (a) surface 1 with t-Boc-protected amino groups, (b) after deprotection to yield free amino groups (surface 2), and (c) after coupling of the heterobifunctional linker SMP to the amino groups (surface 3). The residuals of the fits are shown below the respective spectra.

Figure 2. (a) Route for immobilization of thiols: Aminoterminated surfaces (surface 2) were formed by reaction of t-Boc undecenamine with Si(111)-H to give surface 1 followed by removal of the t-Boc protecting group. Coupling of the heterobifunctional linker SMP to the amino groups introduced maleimide functions onto the monolayer (surface 3) for immobilization of sulfhydryl-containing molecules (surface 4). (b) Thiol addition to surface 3 was investigated using decanethiol, 2,2,2-trifluoroethanethiol, and N-(2-mercaptoethyl)-14C-acetamide.

Figure 3. XP survey spectrum of a monolayers formed by UVlight-mediated hydrosilylation of t-Boc undecenamine with Si(111)-H, yielding surface 1. The inset shows the narrow scan of the silicon 2p region.

protected undecanamine in a UV mediated hydrosilylation reaction. As expected, the 1s peaks of carbon, nitrogen, and oxygen were detectable at 285, 400, and 532 eV and the 2p and 2s peaks of silicon were seen at 99 and 151 eV, respectively. Further, a contamination with residual fluoride ions from the etching procedure was detected at

686 eV. The XP narrow scan of the silicon 2p region showed that only very little silicon dioxide (which would be seen as a broad peak between 102 and 104 eV) was associated with the surface (Figure 3, inset). The level of silicon dioxide varied between samples and was presumably dependent on the purity of the reagents used for etching and hydrosilylation reactions and the reaction conditions.46 It was generally observed that, for high-quality (low-oxide) samples, the deprotection and coupling reactions at the amino-terminated surface, as described below, did not lead to significantly higher silicon dioxide levels. The XP narrow scans of the carbon 1s, nitrogen 1s, and oxygen 1s regions were consistent with the presence of a t-Boc-protected undecanamine monolayer. The carbon 1s region (Figure 4a) exhibited the characteristic binding energies of the O-C-Me3 and N-C(O)-O carbons of the t-Boc protecting group at 287.5 and 289.8 eV, respectively.37 Peak fitting revealed that the binding energies of the amino-alkyl chain were detectable with a large peak at 285.0 eV due to the C-C bonded carbons and a small peak at 286.4 eV, which was assigned to the carbon bonded to the amino group. The nitrogen peak at 400.2 eV (Figure 5a) exhibited a small full-width-half-maximum value (1.2 eV) which indicated that the protected amino-function did not cross-react with the Si-H surface (a broad peak would indicate chemical heterogeneity, which has been observed for monolayers prepared with unprotected amines36). The oxygen 1s narrow scan (Figure S2b, Supporting Information) revealed the presence of at least two distinct binding energies. The peak at 533.5 eV was assigned to the C-O oxygen, and the peak at 532.3 eV was assigned to the CdO oxygen of the t-Boc group. The peak at 532.3 eV was found to have a slightly larger area, presumably due to contributions from the bridging oxygen of silicon dioxide. 47,48 Thermal methods did not yield high-quality t-Bocprotected amino-terminated monolayers in our hands (46) The freshly prepared Si-H surface (etched in CMOS grade 40% NH4F solution and briefly rinsed with MilliQ water) showed no silicon dioxide peak in the silicon 2p narrow scan but was associated with trace levels of adventitious oxygen in the XP wide scan, which may be due to traces of silicon dioxide formed by rinsing with water.35 During the hydrosilylation reaction, oxide formation could result from the presence of traces of water and oxygen, which are known to have a higher reactivity with the hydride-terminated silicon surface than with alkenes.20 (47) Uno, K.; Namiki, A.; Zaima, S.; Nakamura, T.; Ohtake, N. Surf. Sci. 1988, 193, 321-335.

Characterization of Organic Multilayers on Si(111)

Figure 5. XP narrow scans of the nitrogen 1s signal of (a) surface 1 with t-Boc-protected amino groups, (b) after deprotection to yield surface 2, and (c) after coupling of the heterobifunctional linker SMP to the amino groups (surface 3). The residuals of the fits are shown below the respective spectra.

using various conditions. Monolayers prepared by immersion of the Si-H sample into t-Boc undecenamine at 100 °C49 for 16 h were associated with high levels of silicon dioxide. Relatively high silicon dioxide formation was also observed for samples prepared in a refluxing mesitylene solution of the alkene, although t-Boc undecenamine appeared to be stable (no decomposition detected by 1H NMR spectroscopy) under these reaction conditions. Removal of the Protecting Group and Reaction with SMP. The deprotection of the amino group and the formation of the amide could be followed in the carbon 1s signal. After removal of the t-Boc group with TFA, the binding energies characteristic of the t-Boc group disappeared37 (Figure 4b). The remaining signal consisted of two components corresponding to the carbons of the alkyl chain at 285.0 eV and the carbon adjacent to nitrogen at 286.4 eV. The ratio between these peaks was approximately 10:1, which was consistent with the undecanamine layer. Linking of the heterobifunctional linker SMP to the free amino groups was confirmed by the appearance of a new peak centered at 288.6 eV due to the binding energies of the CdO carbons of the amide group and the imide groups of the maleimide moiety after the coupling reaction (Figure 4c). The carbon envelope was fitted with four components at 285.0, 286.5, 288.0, and 288.9 eV. The peak at 285.0 eV, which was assigned to the C-C carbons of the alkyl chain, was slightly broadened (full-widthhalf-maximum 1.4 eV) because it also contained contributions from the CdC carbons and the carbon adjacent to the amide group in the linker molecule. The peak of the C-N binding energy at 286.5 eV was slightly increased compared to the amino-terminated layer as a result of the presence of the linker (which also contains a C-N carbon). In agreement with the range of binding energies reported in the literature, the peak of the amide carbon occurred at 288.0 eV, while the imide carbons were seen at 288.9 eV. It was apparent that the derivatization of the aminoterminated surface with SMP did not go to completion under the conditions used for preparation of the sample shown in Figure 4c. On the basis of peak areas it was estimated that approximately one SMP molecule was bound per two alkyl chains (50% yield). A lower yield of approximately 30% for the coupling of SMP to (3(48) The nonbridging oxygen of silicon dioxide would appear at a similar binding energy as the peak assigned to the C-O oxygen.47 Consequently, this peak might also have a silicon dioxide component, which cannot be quantified accurately. (49) The t-Boc protecting group was not stable at 200 °C. Monolayer formation at this temperature resulted in samples with high oxide levels, presumably as a result of side reactions of the decomposition products (such as the deprotected amine) with the Si-H surface.

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aminopropyl)triethoxysilane SAMs on titanium has been reported.50 The authors suggested that steric hindrance would be an important factor precluding higher yields especially for the short chain linker SMP which would be less flexible than linker molecules with longer chains. Differences in the derivatization yields reported in the literature50,51 and those observed here may be related to the accessibility of the amino groups on the surface, which may be dependent on the surface roughness of the substrate.51 Similarly, these steps could be followed in the nitrogen 1s signal (Figure 5). The nitrogen peak of surface 3 had a binding energy of 400.2 eV and exhibited a small fullwidth-half-maximum value (1.2 eV). After deprotection, the nitrogen signal showed two peaks. The main peak at 400.0 eV corresponded to the unprotonated amino group (-NH2) while the minor peak at 401.7 eV was due to the protonated amino group (-NH3+). The nitrogen peak after linking of SMP to the surface occurred at 400.5 eV and was broader (full-width-half-maximum 1.6 eV) since it contained contributions from the chemically slightly different nitrogens of the amide linkage, the maleimide group, and unreacted amino groups. Removal of the relatively hydrophobic t-Boc protecting group was further confirmed by contact-angle measurements. The contact angle of surface 1 with water was 76 ( 4°, which decreased progressively with the time of immersion in 40% TFA in dichloromethane to 66 ( 3° after 10 min, 60 ( 1° after 15 min, and 56 ( 2° after 20 min. The value after 20 min was in good agreement with literature data for a deprotected, amino-terminated monolayer.37 Immobilization of Thiols on Maleimide-Modified Surfaces. Sulfur was not detectable in XP survey scans of maleimide-derivatized surfaces reacted with a variety of thiols. Consequently, a thiol with a trifluoromethyl group was immobilized on the surface since this group could be detected with higher sensitivity than sulfur by XPS. XP survey scans of surface 3 reacted with 2,2,2trifluoroethanethiol showed the presence of the fluorine 1s peak, along with the expected signals for silicon, carbon, nitrogen, and oxygen. Two signals were present in the narrow scan of the fluorine 1s region (Figure 6b). The large peak at 688.8 eV was assigned to the trifluoromethyl group and the small peak at 686.0 eV to contaminating fluoride ions remaining on the surface after preparation of the Si-H surface. The contamination with fluoride ions was already detected for the Si-C linked t-Boc protected monolayer (surface 1, Figure 6a). The carbon signal showed the peak of the trifluoromethyl carbon at 293.4 eV, the carbonyl carbons of the amide linkage and the maleimide group at 288.9 eV, and the remaining carbons as a broad peak at 285.0 eV with a shoulder on the high binding energy side due to the carbons attached to nitrogen. The peak ratios between the component peaks belonging to the alkyl chain, the linker molecule, and the thiol indicated that neither the derivatization with SMP nor the coupling of the thiol to the maleimide group had gone to completion. N-(2-Mercaptoethyl)-14C-acetamide was synthesized to serve as a radioactively labeled probe for the detection and quantification of thiol binding on surface 3. The 14Clabeled probe was reacted with a freshly prepared maleimide-functionalized surface. On a control surface, the (50) Xiao, S.-J.; Textor, M.; Spencer, N. D.; Sigrist, H. Langmuir 1998, 14, 5507-5516. (51) Rezania, A.; Johnson, R.; Lefkow, A. R.; Healy, K. E. Langmuir 1999, 15, 6931-6939.

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Figure 6. XP narrow scans of the fluorine 1s region of (a) Si(111) derivatized with a t-Boc-protected undecanamine monolayer (surface 1) and (b) after deprotection, modification with SMP, and reaction with 2,2,2-trifluoroethanol.

maleimide groups were reacted with 2-mercaptoethanol prior to immersion in a solution of the probe molecule. After removal of physisorbed material by sonication, the radioactivity remaining on the surfaces was determined by scintillation counting. The 14C count on the maleimidederivatized samples was approximately four times higher than on control samples. This provides further evidence for the binding of thiols to maleimide groups on surface 3. The density of the probe on the surface (determined after subtraction of the 14C count measured on the control sample) was estimated to be 0.1-0.2 nmol cm-2, which corresponded to a ratio of bound, radioactively labeled thiols to alkyl chains in the monolayer of approximately 1:3-1:7.52 The surface concentration determined here was within the range determined for the binding of 35S-labeled cysteine to SMP derivatized (3-aminopropyl)triethoxysilane monolayers on titanium surfaces (0.04-0.71 nmol cm-2).50 X-Ray Reflectometry of Reactions on AminoTerminated Monolayers. Observed and fitted X-ray reflectivity data as a function of momentum transfer (Qz) for a series of surfaces formed by reactions at aminoterminated monolayers are shown in Figure 7. The measured reflectivity curves of these films varied over seven orders of magnitude before the background was reached. Only one minimum was present in the reflectivity data because of the ultrathin nature of the films in this study. Minima of fringes in these types of reflectivity curves move to lower Qz as the overall thickness of the structure increases. Examination of Figure 7 clearly shows that the stepwise modification of the surface and assembly of the multilayer structures could be followed by X-ray reflectivity. Reflectivity data were fitted using both monolayer and multilayer structural models. In the modeling process, the electron density of the silicon substrate was fixed at 0.80 e- Å-3. However, the roughness of the silicon/film interface was determined by refining the fits. For the t-Bocprotected undecanamine film, an adequate fit was achieved using a monolayer structural model. A multilayer structural model for this surface did not improve the fit despite the expected difference in electron density between the (52) This ratio was estimated on the basis of an area per molecule of 25 Å2 for the alkyl chains, which corresponds to a surface concentration of 0.66 nmol cm-2.

Bo¨ cking et al.

Figure 7. X-ray reflectivity curves of modified Si(111) surfaces: (a) t-Boc-protected undecanamine monolayer (surface 1), (b) deprotected (free) undecanamine monolayer (surface 2), (c) undecanamine monolayer after reaction with SMP (surface 3), and (d) maleimide-modified surface after reaction with decanethiol (surface 4). The solid lines represent model fits to the reflectivity data. Curves b, c, and d have been offset for clarity. Table 1. Refined Structural Parameters for Engineered Thin-Film Heterostructures on Amino-Terminated Surfacesa 1

2

3

4

Thiol Layer d (Å) Fel (e- Å-3) σ (Å)

6(1) 0.28(1) 3(1) Maleimide Layer

d (Å) Fel (e- Å-3) σ (Å) d (Å) Fel (e- Å-3) σ (Å)

16(1) 0.40(1) 2(1)

σ (Å)

3(1)

Base Layer 13(1) 0.32(1) 2(1) Silicon 3(1)

5(1) 0.46(1) 3(1)

5(1) 0.50(1) 2(1)

12(1) 0.32(1) 2(1)

12(1) 0.32(1) 3(1)

3(1)

4(1)

a

t-Boc-protected undecanamine monolayer (surface 1), undecanamine monolayer (surface 2), after reaction with SMP (surface 3), and after reaction with SMP and decanethiol (surface 4).

alkyl layer and the electron-rich t-Boc terminal group. Multilayer models were required to fit the XR data obtained after the coupling reactions at the aminoterminated surface. In the case of the heterogeneous structure formed after linking of SMP (surface 3), a twolayer model was required to give the best fit to the data, while a three-layer model was required for the structure after immobilization of decanethiol on the SMP-derivatized undecanamine monolayer (surface 4). The refined structural models (thickness, film density, and interfacial roughness) based on these fitted reflectivity data are given in Table 1. The removal of the t-Boc group reduced the apparent thickness from 16(1) Å (surface 1) to 13(1) Å (surface 2), which is evident from the shift in the reflectivity minimum to higher Qz (Figure 7a and b). The reduced thickness of the amino-terminated alkyl monolayer (compared to the length of the fully extended chain) may be the result of gauche defects and/or a tilt of the chains from surface normal. After attachment of the heterobifunctional linker SMP, a second layer of 5(1) Å thickness was attached to a 12(1) Å thick base layer. In the final derivatization step, decanethiol was linked to the surface-immobilized ma-

Characterization of Organic Multilayers on Si(111)

leimide groups. The resulting 6(1) Å thick alkanethiol layer was approximately half as thick as would be expected for a fully extended chain which indicated that the layer had collapsed. This could be due to an incomplete reaction of the amino groups of surface 2 with SMP as a result of steric hindrance, resulting in missing binding sites for the formation of a dense alkanethiol layer. The refined electron densities of the undecanamine monolayer (as well as those of the alkyl sublayers of the multilayer structures) were found to be 0.32(1) e- Å-3. This value is in excellent agreement with average electron density of 0.31 e- Å-3 for a series of alkyl monolayers on Si(100) and Si(111)25 and the electron density of 0.30 eÅ-3 for a monolayer prepared from 50% heptadecene and 50% diheptadecanoyl peroxide on Si(111)-H.24 Considering that the electron density of crystalline paraffin, C33H68, is 0.35 e- Å-3,53 the value indicated a densely packed monolayer with some defects. As expected, the electron density of the t-Boc-protected undecanamine layer was higher as a result of the electron-rich protecting group. To facilitate the comparison between the t-Boc-protected and the free undecanamine monolayers, one may determine the area per molecule, A, from the reflectivity data,54 which provided an estimate of the molecular coverage of the silicon surface. The area per molecule for the monolayer with protected amino groups ((24 ( 2) Å2/molecule) was in good agreement with that of the monolayer with free amino groups ((23 ( 3) Å2/molecule). The area per molecule for the linker layer was ∼35-40 Å2/molecule, which corresponded to a ratio of approximately one linker molecule per 1.5-1.7 alkyl chains. For the decanethiol layer, the area per molecule (∼50 Å2/molecule) gave an estimate of approximately one decanethiol molecule bound per two alkyl chains. The roughness (determined by refining the fits) of the substrate/monolayer interfaces was within expectations for the silicon surface. In a number of instances, the outside of a given monolayer was observed to be slightly smoother than the substrate/monolayer interface. The ability of these types of monolayer films to reorganize and form a smoother outer surface has been observed by a number of other researchers.25,55,56 NHS Ester-Terminated Monolayers. NHS esters have been used extensively for the conjugation of carboxylic acids and amines in solution, as well as for immobilization of biological molecules on surfaces due to their relatively high stability in aqueous media.57 Proteins could couple to NHS ester surfaces mainly via lysine side chains and the amino terminus. The advantage of preparing reactive, ester-terminated surfaces is that no further activation steps are required for the immobilization of nucleophiles. N-Succinimidyl undecenoate was attached to the silicon surface in a thermal or UV-light-mediated hydrosilylation reactions as shown in Figure 8. 4-(Trifluoromethyl)benzylamine was subsequently reacted with the monolayer to demonstrate the immobilization of amines, which was characterized by XPS and XR. (53) Ewen, B.; Strobl, G. R.; Richter, D. Faraday Discuss. Chem. Soc. 1980, 69, 20-31. (54) The area per molecule is given by A ) Ne/(dFel), where d is the measured film thickness and Ne is the number of electrons per molecule in the monolayer. (55) Sieval, A. B.; Opitz, R.; Maas, H. P. A.; Schoeman, M. G.; Meijer, G.; Vergeldt, F. J.; Zuilhof, H.; Sudhoelter, E. J. R. Langmuir 2000, 16, 10359-10368. (56) Asmussen, A.; Riegler, H. J. Chem. Phys. 1996, 104, 81518158. (57) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, 1996.

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Figure 8. (a) Route for the immobilization of amines: Modification of Si(111)-H with N-succinimidyl undecenoate gave NHS ester-terminated monolayers (surface 5), which were subsequently reacted with amines (surface 6). (b) The aminolysis reaction was investigated using 4-(trifluoromethyl)benzylamine.

Figure 9. (a) XP survey spectrum of an NHS ester-terminated monolayer on Si(111) (surface 5). (b) After reaction of the NHS esters with 4-(trifluoromethyl)benzylamine. The insets show the silicon 2p narrow scans of the respective surfaces.

Characterization of NHS Ester-Terminated Surfaces and Amine Binding by XPS. Figure 9a shows the XP survey spectrum of such a monolayer prepared by immersing the freshly etched silicon wafer in the neat alkene at 95 °C for 16 h. Peaks corresponding to the carbon 1s, oxygen 1s, and nitrogen 1s binding energies were found on the sample at 285, 532, and 400 eV, respectively, as expected for the elemental composition of the molecular layer. The sample shown also contained a contamination with residual fluoride from the etching procedure as evident from the peak at 686 eV. The XP narrow scans of the carbon 1s and oxygen 1s regions (Figure S3, Supporting Information) were consistent with literature data of porous silicon modified by immersion in a solution of the alkene in heptane at 120 °C.58 The carbon signal was deconvoluted

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into three components. The high-binding-energy peak (289.6 eV) was assigned to the carbonyl groups of the ester and the NHS moiety, while the peak at 286.3 eV was assigned to the carbons alongside the carbonyls. The remaining carbons of the alkyl chain (C-C) appeared at 285.0 eV. The ratio between these peaks was approximately 1:1:3, which was consistent with the structure of the compound. Three peaks were fitted to the oxygen signal. The minor peak at 535.4 eV was assigned to the C-O-N oxygen, and the major peak at 533.1 eV was mainly due to the CdO oxygens of the NHS ester.59 Finally, a small peak consistent with the binding energy of bridging oxygen in silicon dioxide was present at 531.9 eV.60 The binding energy of the nitrogen of the NHS group was 402.3 eV. The presence of the electronegative oxygen on the nitrogen is consistent with the observed shift by 2 eV.59 In a number of samples, the XP narrow spectrum of the nitrogen 1s region also showed a minor nitrogen species with a binding energy of 400.4 eV, which was presumably due to contamination of the surface. High-quality mixed monolayers with very low oxide levels could also be formed by attachment of a mixture of N-hydroxysuccinimidyl undecenoate and decene in a 1:2 molar ratio in refluxing mesitylene. XPS analysis of these samples confirmed the presence of the NHS moiety by the characteristic nitrogen 1s signal at 402.3 eV. Silicon dioxide was not detectable in significant levels in the narrow scan of the silicon 2p region. The third method pursued for the formation of surface 5 involved covering the Si(111)-H surface with a solution of the N-hydroxysuccinimidyl undecenoate in mesitylene and irradiation with UV light. The resulting monolayers showed the characteristic nitrogen peak of the NHS ester but were associated with detectable oxide levels as evident by the peak at 103 eV in the XP narrow scan of the silicon 2p region. Immobilization of amines on the NHS ester-terminated monolayer was confirmed by XPS. The coupling of 4-(trifluoromethyl)benzylamine was evident in the survey spectrum by the appearance of a large fluorine peak (Figure 9b). In the fluorine 1s narrow scan, the peak of the trifluoromethyl group appeared at 688.4 eV. In addition, a small peak at 686.0 eV was also detectable, indicating the presence of a contamination with fluoride ions from the etching procedure. The narrow scan of the silicon 2p region after the surface reaction showed that the levels of silicon dioxide were not significantly increased. Figure 10 shows the nitrogen 1s narrow scans of the activated ester surface and the surface after reaction with 4-(trifluoromethyl)benzylamine. Loss of the nitrogen 1s binding energy at 402.3 eV suggested that the NHS moiety was removed from the surface as a result of aminolysis and possibly competing hydrolysis reactions. Instead, the nitrogen signal showed a peak at 400.2 eV, which was consistent with the formation of an amide linkage with the amino group of 4-(trifluoromethyl)benzylamine. The complete removal of the NHS moiety was further confirmed by the loss of the oxygen peak at 535.4 eV, which was characteristic to the C-O-N oxygen. The carbon 1s signal displayed a broad peak centered at 284.8 eV with components due to the alkyl chain, the aromatic carbons (58) Wojtyk, J. T. C.; Morin, K. A.; Boukherroub, R.; Wayner, D. D. M. Langmuir 2002, 18, 6081-6087. (59) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (60) Since the ratio between the CdO oxygen and C-O-N oxygen peaks was 4:1, which was larger than the expected ratio of 3:1, it appeared likely that the peak at 533.1 eV also contained nonbridging oxygen from silicon dioxide.47

Bo¨ cking et al.

Figure 10. XP narrow spectra of the nitrogen 1s region of (a) the NHS ester-terminated surface and (b) after reaction with 4-(trifluoromethyl)benzylamine. The loss of the nitrogen peak of the NHS ester at 402.3 eV and the concomitant appearance of a nitrogen peak at 400.2 eV indicated the conversion of the NHS ester moieties to amides.

Figure 11. X-ray reflectivity curve for Si(111) surfaces modified with a monolayer of N-succinimidyl undecanoate after aminolysis of the NHS esters with 4-(trifluoromethyl)benzylamine (surface 6). The solid line represent a model fit to the reflectivity data.

(shifted to lower binding energies), and the carbon attached to nitrogen (shifted to higher binding energies). Further, the peak of the trifluoromethyl carbon and the carbonyl carbon of the amide group were present at 292.7 and 288.0 eV, respectively. Characterization of Amine Binding by XR. Derivatization of the NHS ester-terminated surface could also be followed by XR. Observed and fitted reflectivity data for the surface after aminolysis with 4-(trifluoromethyl)benzylamine are shown in Figure 11. A two-layer model consisting of a 10(1) Å thick alkyl base layer and a 6(1) Å thick top layer was required to fit the data. The electron density of the alkyl base layer was 0.32(1) e- Å-3, which was identical to that observed for the aminoterminated monolayer described above. The top layer had an electron density of 0.57(2) e- Å-3 and was attributed to electron-rich 4-(trifluoromethyl)benzylamine molecules immobilized on the monolayer surface. Conclusions Two strategies for the immobilization of molecules on Si-C linked ω-functionalized monolayers on silicon

Characterization of Organic Multilayers on Si(111)

substrates were investigated using model compounds. In the first strategy, a range of thiol probes were linked to amino-terminated monolayers via heterobifunctional linkers. In the second strategy, amines were immobilized on monolayers terminated with activated NHS ester moieties. Reaction of Si-H surfaces with t-Boc-protected undecenamine yielded 16 Å thick monolayers. The removal of the protecting group with trifluoroacetic acid was evident from a decrease in the layer thickness by 3 Å and the disappearance of the characteristic binding energies of the t-Boc protecting group in the carbon 1s region of the XP spectra. The electron density of the resulting aminoterminated layer was consistent with a densely packed layer. Reaction of the terminal amino groups with the heterobifunctional linker SMP introduced a 5 Å thick linker layer onto the monolayer. The reactivity of the surface-immobilized maleimide groups was demonstrated with a range of thiol probes. The surface density of binding sites for thiols was found to be approximately 0.1-0.2 nmol cm-2, which corresponded to a ratio of thiol-to-alkyl chains of 1:3 to 1:7. In conclusion, reactions occurred with high efficiency on the amino-terminated surface, and no significant degradation of the quality of the layer occurred during the deprotection and coupling reactions in organic and aqueous solutions. Hence, the method presented here could be used for the site-specific immobilization of proteins, peptides, and carbohydrates for the fabrication of biochips with increased sensitivity.9

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NHS esters were introduced onto the silicon surface by reaction of the Si-H surface with N-succinimidyl undecenoate. Assembly of a covalently linked layer of 4-(trifluoromethyl)benzylamine on top of the Si-C linked monolayer by reaction with the terminal NHS ester groups was confirmed by XPS and XR. Acknowledgment. The authors thank Christopher Chidsey and Danial Wayner for helpful suggestions for the preparation of Si-C linked monolayers. The authors acknowledge the assistance of Ian Gentle and Jeremy Ruggles (Brisbane Surface Analysis Facility, University of Queensland) for help with the X-ray reflectometry measurements and Bill Gong for help with the XPS measurements. Funding was provided by the UNSW Faculty Research Grants Program. T.B. was supported by an International Postgraduate Research Scholarship, a UNSW Postgraduate Award, and an AINSE Postgraduate Research Scholarship. Supporting Information Available: The syntheses of the functionalized alkenes, the heterobifunctional linker SMP, and the radioactively labeled probe N-(2-mercaptoethyl)-14Cacetamide and additional XP spectra. This material is available free of charge via the Internet at http://pubs.acs.org. LA048474P